Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/35—Mycoplasma

Definitions

• the present invention relates to an attenuated vaccine strain of Mycoplasma hyopneumoniae, a live vaccine containing the attenuated strain, and the use thereof for protecting a disease associated with Mycoplasma hyopneumoniae infection.
• Mycoplasma hyopneumoniae commonly known as swine asthma, is a chronic respiratory tract infection caused by Mycoplasma hyopneumoniae (Mhp). The main symptoms are cough and asthma. The disease mainly causes the pig's feed rate to decrease and growth to be blocked, with high incidence and low mortality. Mycoplasma pneumoniae is widespread worldwide and causes significant economic losses to the modern pig industry.
• Mycoplasma hyopneumoniae often forms a mixed infection with other pathogens.
• Pasteurella Multocida Streptococcus Suis (SS), Haemophilus Parasuis of Swine (HPS) or Actinobacillus Pleuropneumonia of Porcine (APP) Such as mixed infection, causing local epidemic pneumonia in pigs.
• RIRSV Porcine Reproductive and Respiratory Syndrome
• PCV2 Porcine Circovirus Type 2
• SIV swine influenza virus
• PRDC Porcine Respiratory Disease Complex
• Mycoplasma pneumoniae is an intractable chronic infectious disease.
• the effect of drug treatment is not ideal. It is easy to relapse after stopping the drug. Once the disease is popular in the farm, it is difficult to eradicate. Therefore, the prevention, control and purification of Mycoplasma hominis pneumonia requires comprehensive measures such as early diagnosis, timely isolation, antibiotic treatment or elimination of sick pigs, vaccine prevention, total access, strict disinfection, etc., in which vaccine prevention starts in the control of Mycoplasma pneumoniae A key role.
• Mycoplasma hyopneumoniae binds to bronchial ciliated epithelial cells via respiratory tract infection to form colonization, resulting in persistent infection.
• Inactivated vaccines stimulate a systemic immune response, and the effect of inducing a local immune response is poor.
• the circulating antibodies produced by the inactivated vaccine must pass through the epithelial barrier and secrete into the tracheobronchial lumen, which is extremely difficult and inefficient.
• the vaccine In order to achieve effective immunity, the vaccine must be able to stimulate a high level of systemic immune response. Of course, this requires a relatively high antigen dose and a strong adjuvant, which makes these vaccine costs more expensive.
• the immune response produced by the inactivated vaccine can only inhibit the proliferation of wild-type infection and reduce the degree of tissue damage, and can not completely prevent the infection of wild-type virus, so it is difficult to achieve ideal control of the disease. Summary of the invention
• the object of the present invention is to provide a live attenuated vaccine against Mycoplasma hyopneumoniae, which can be used for the prevention and control of diseases caused by Mycoplasma hyopneumoniae infection, including diseases caused by co-infection of other pathogenic bacteria with Mycoplasma hyopneumoniae.
• the live attenuated vaccine of Mycoplasma hyopneumoniae is different from the inactivated vaccine. After inoculation, it can proliferate in the body and effectively activate the body's immune system. At the same time, the live vaccine can colonize the bronchial ciliated cells after entering the respiratory system, resulting in a "occupation". The effect "can make the wild poison unable to colonize and block the infestation process of wild poison from the source, thus playing a good anti-infective effect. At the same time, because live vaccines can proliferate in vivo and effectively activate the immune system, the required dose is much lower than inactivated vaccines, so it has obvious advantages over inactivated vaccines in cost control, which is conducive to saving social resources.
• the key to the development of live vaccines is the weakening of the strains that increase the virulence of the strains while maintaining the immunogenicity of the highly immunogenic strains.
• the present invention uses the first generation of the rabbit lungs to subculture, and after 100 passages, the cell-free medium is subdivided.
• the method has the advantages that the in vivo passage of the animal in the early passage process can avoid the defect that the strain is easily weakened in the early stage and is not conducive to the maintenance of immunogenicity when the medium is used in vitro.
• Subsequent use of cell-free medium for subsequent generation is beneficial to reduce the weaker generation and shorten the weakening process to a certain extent.
• the specific source of the strain is: Screening with typical Mycoplasma hyopneumoniae infection without obvious other pathogens
• the infected pig lung disease material was subcultured to the 100th generation using the lung of the rabbit, and then the M. hyopneumoniae strain was isolated and serially passaged by the medium, and simultaneously obtained by screening of a plurality of strains.
• the selected strain was named as Mycoplasma hyopneumoniae (Mhp) attenuated strain AN306. After being weakened for a long time, the attenuated strain loses its virulence, is safe to animals, and retains good immunogenicity.
• the attenuated strain AN306 can be used in the preparation of a Mycoplasma hyopneumoniae pneumonia vaccine or a multiple vaccine.
• the attenuated strain of Mycoplasma hyopneumoniae was deposited on October 25, 2012 in the China Center for Type Culture Collection in Wushan, Wuchang, Wuhan, under the accession number CCTCC M 2012431.
• Another object of the present invention is to disclose the live attenuated vaccine against Mycoplasma hyopneumoniae, which can be a Mycoplasma hyopneumoniae pneumonia vaccine or a multiple vaccine.
• the above-mentioned Acoplasma hyopneumoniae (Mhp) attenuated strain AN306, and a pharmaceutically acceptable carrier or adjuvant are included.
• Carriers or excipients mainly include three types of substances, which vary depending on the specific use state of the vaccine and the route of immunization.
• One type is solvent or buffer system, including sterile physiological saline, sterile phosphate buffer; one type is lyoprotectant, including skim milk powder, gelatin, sucrose, when the vaccine is prepared in lyophilized form, it needs to be added to lyophilized
• the system helps the live vaccine to maintain activity; the first type is a spray protectant, mainly glycerin.
• a spray protectant mainly glycerin.
• the live vaccine formulation of Mycoplasma hyopneumoniae of the present invention may further comprise an immunological adjuvant.
• Adjuvants are a common ingredient in vaccine formulations and are used to enhance the immune stimulating ability of the vaccine itself.
• the adjuvant used in the present invention is an adjuvant suitable for a live vaccine against Mycoplasma hyopneumoniae, i.e., does not cause significant damage to the viability of the live vaccine, and is suitable for the immune requirement of Mycoplasma pneumoniae.
• the immunoadjuvant disclosed by the invention may be a single adjuvant component or a composite of a plurality of adjuvant components, and the adjuvant component may include a Chinese herbal polysaccharide, a carbomer, a chitosan, an immunostimulating complex matrix, One or more of levamisole, dextran, CpG, and capsular.
• the various immunoadjuvant ingredients, ratios, and dosages used will vary depending on the particular dosage and route of administration of the live vaccine.
• the live vaccine preparation of Mycoplasma hyopneumoniae pneumoniae of the present invention may further comprise at least one immunogen of another pathogen, and is formed into a joint vaccine for use.
• Other pathogens include at least one of viruses, bacteria, fungi, and parasites, such as porcine circovirus, Haemophilus parasuis, porcine reproductive and respiratory syndrome virus, pig nose Mycoplasma, Mycoplasma hyopneumoniae, swine influenza virus, etc. These pathogens often form mixed infections or secondary infections in the respiratory tract with M. hyopneumoniae in the clinic, which promote each other, leading to aggravation of the disease and posing a major threat to the health of the pig population.
• the combination of these pathogen vaccines with a live vaccine against Mycoplasma hyopneumoniae can be used to prevent mixed infections of Mycoplasma pneumoniae and other pathogens.
• the immunogen of the other pathogens may be in the form of live bacteria or live poison, or may be in an inactivated form, a protein subunit form or the like. It is worth noting that the carriers, excipients and adjuvants used in other pathogen vaccines must be compatible with the system used in the live vaccine of Mycoplasma hyopneumoniae and must be tolerated for this live vaccine.
• the live attenuated vaccine against Mycoplasma hyopneumoniae of the present invention can be immunized by various routes including intrapulmonary injection, intramuscular injection, aerosol immunization, oral immunization and intranasal immunization.
• the preparation for the live attenuated vaccine may be an intrapulmonary injection, an intramuscular injection, an aerosol, an oral preparation or a nasal drop.
• the choice of immunization route depends on the animal to be immunized, the immunization history, the farm environment, and the ease of operation of the vaccination personnel.
• Mycoplasma hyopneumoniae infects the body and first binds to the bronchial epithelial ciliated cells of the lungs, causing the cilia to fall off, which in turn infects the tissue, induces inflammation, and opens the door for infection by other pathogens.
• the live vaccine of Mycoplasma hyopneumoniae When the live vaccine of Mycoplasma hyopneumoniae is immunized by intrapulmonary injection, the live vaccine can be directly injected into the target tissue, and bind to the ciliated cells, thereby occupying the binding site and preventing the infection of wild poison; when the live vaccine of Mycoplasma hyopneumoniae is immunized by aerosol By controlling the particle size of the droplets, the live vaccine can be allowed to reach the lower respiratory tract of the animal, effectively binding to the bronchial ciliated cells, and the use of an aerosol vaccine in a closed barn or space can reduce the number of personnel required for immunization; Intramuscular injection is the most commonly used method of inoculation for animal immunization.
• the live vaccine of Mycoplasma hyopneumoniae When the live vaccine of Mycoplasma hyopneumoniae is immunized by intramuscular injection, it can induce systemic cellular and humoral immune responses in animals. Partial target tissue can inhibit the infection of Mycoplasma hyopneumoniae wild venom; When the live vaccine of Mycoplasma hyopneumoniae is inoculated by oral or intranasal, it can induce intestinal mucosal immune response in the intestine or nasal cavity, through the common mucosal immune system, lymphocytes Can homing to different mucosal sites, thus achieving The immune response of the bronchopulmonary tissues of the respiratory tract, to protect against the infection of wild mycoplasma pneumoniae.
• the selected immunological adjuvant will also vary depending on the route of inoculation used. Therefore, the live attenuated vaccine of Mycoplasma hyopneumoniae of the present invention can be applied to the prevention and treatment of Mycoplasma hyopneumoniae infection.
• the live attenuated vaccine against Mycoplasma hyopneumoniae of the present invention is prepared by using Mycoplasma hyopneumoniae (Mhp) attenuated strain AN306 with a pharmaceutically acceptable carrier or adjuvant, and at least one immunological adjuvant, at least one other pathogen. The immunogens are combined.
• Mhp Mycoplasma hyopneumoniae
• the live vaccine of Mycoplasma hyopneumoniae of the present invention may be administered in a single immunization, or two or more repeated inoculations may be used to enhance the immune response. It is specifically selected depending on the route of immunization and the condition of the animal.
• the single immunization time is usually 3-10 days old, preferably 5-7 days old.
• the first immunization time for two or more repeated inoculations is usually 3-10 days old, preferably 5-7 days old.
• the time interval between two immunizations is typically 2-3 weeks.
• Biological sample preservation information is typically 3-10 days old, preferably 5-7 days old.
• Mycoplasma hyopneumonia AN306 Mycoplasma hyopneumonia AN306 (Mycoplasma hyopneumoniae AN306) was deposited on October 25, 2012, at the China Center for Type Culture Collection in Wushan, Wuchang, Wuhan, with the accession number CCTCC. M 2012431. Specific examples:
• the present invention also verifies the live vaccine formulation of Mycoplasma hyopneumoniae pneumoniae, including a plurality of different immunization routes, A variety of different adjuvants, adding other different pathogen immunogens, can effectively prevent infection of Mycoplasma hyopneumoniae, control the occurrence of Mycoplasma pneumoniae, special experiments and preparations.
• Example 1 Acquisition of Mycoplasma hyopneumoniae AN306
• the selected 10 virulent tissue poisons were inoculated into local breeds of healthy susceptible pigs by intranasal drip, after challenge
• the clinical symptoms of Mycoplasma hyopneumoniae such as cough, asthma, weight loss, and canine sitting were observed in the test animals.
• typical shrimp-like changes were observed in the sharp leaves and heart leaves of the lungs.
• clinical symptoms, incidence ratio, lung lesion degree and serum agglutination antibody titer of animal challenge the three most virulent and immunogenicity were selected from 10 virulent tissue poisons for passage. Passage weakened:
• the virulent tissue was injected into the lungs of healthy rabbits, and the lungs of the rabbits were collected. After homogenization, the healthy rabbits were inoculated and subcultured. Only one of the above three virulent strains was successfully passaged in rabbits. The strains were divided into 20 generations, 40 generations, 60 generations, and 80 generations, and each of them was divided into successive passages and successive passages to 100 generations.
• Mycoplasma hyopneumoniae was isolated from the lungs of 100 generations of lactating rabbits using the "sick lung immersion method".
• the specific method is as follows: animal bloodletting death, aseptic operation of collecting lung tissue, cutting 1/2 sesame-sized pieces, washing with Hank's solution in KM2 medium, adding antibiotics, culturing at 37 ° C, daily observation and culture pH change and turbidity of the liquid.
• the pH showed a significant change (down to 7.0-6.8) and there was uniform turbidity (about 48 h)
• the above cultures were serially passaged for 1:5 to 5 inoculation with 1:5 inoculum and then subcultured with 1:10 inoculum. Press
• the isolated microbial cells are polymorphic, with typical morphological characteristics of Mycoplasma hyopneumoniae.
• a microagglutination reaction and a metabolic inhibition test were carried out using Mycoplasma hyopneumoniae standard positive serum, and identified as Mycoplasma hyopneumoniae.
• the isolated M. hyopneumoniae was purified by three solid clones and identified again and stored lyophilized.
• the freeze-preserved F100-derived Mycoplasma hyopneumoniae strain was resuscitated, and serial passage was weakened by using KM2 medium at 1:10. Each five generations were identified by PCR and stored frozen every 20 generations. From F100,
• AN301 F240 grows well, 10 9 CCU/mL no apparent lesions, no pathological changes
• AN302 F110 grows slowly, 10 2 CCU/mL - media passage lost
• AN303 F280 grows well, 10 8 CCU/mL no apparent lesions, no pathological changes
• AN304 F240 grows well, 10 7 CCU/mL no apparent lesions, no pathological changes AN305 F32 suckling rabbits are lost
• AN306 F280 grows well, 10 y CCU/mL no apparent lesions, no pathological changes
• AN307 F140 grows well, 10 8 CCU/mL no apparent lesions, mild inflammatory pathology
• AN308 F300 grows well, 10 9 CCU/mL no apparent lesions, no pathological changes
• AN309 F360 grows well, 10 8 CCU/mL no apparent lesions, no pathological changes
• AN311 F260 grows slowly, 10 4 CCU/mL has no apparent lesions, no pathological changes
• the strains screened above were subjected to immunogenicity assay and challenge immunoprotective assay. Lyophilized bacteria of different strains were prepared, and the titer of each lyophilized strain was determined. The freeze-dried vaccine was dissolved in a sterile phosphate buffer, and each strain was adjusted to the same concentration with a sterile phosphate buffer.
• the immunoprotective power of the 5-7 day old healthy negative Meishan pig was determined. Animals were randomized into groups of 5 each. In the immunized group, 0.5 mL (10 5 CCU/head) of attenuated attenuated strain was injected into the lung. After 8 weeks of immunization, intratracheal injection of Mycoplasma hyopneumoniae was highly virulent, and 28 days later, the necropsy was performed. A healthy control group and a challenge control group were also set up.
• Serum antibodies were measured by indirect hemagglutination method and were judged to be positive by 1:10"++" or more.
• Anatomy score Animals were sacrificed 28 days after challenge, and lung injury in the experimental pigs was scored according to the method reported by MADEC and KOBISCH (1982). The entire lung is divided into left lobe (LCL), left lobe (LAL), left temporal lobe (LDL), right heart (RCL), right lobes (RAL), right temporal lobe (RDL) and accessory cotyledons (IL). ), a total of 7 lung lobe. The damage score for each lung was the sum of the dorsal and ventral lesion scores of the above seven lung lobe, with a total score of 28 points.
• the AN306 attenuated strain has better immunogenicity, and the attacking test proves that it has the highest protective power. And the above test proves that the growth in vitro is fast, the titer is high, and the safety is good. Therefore, the AN306 strain is selected as the live vaccine strain, and the initial generation is F280.
• Example 2 Culture of attenuated strain of Mycoplasma hyopneumoniae AN306 strain
• KM2 liquid medium preparation (according to 2050mL): Eagles solution 1000 mL, hydrolyzed milk protein 10 g, pig serum 400 mL, fresh yeast leach juice 20 mL, Dulbecco phosphate buffer 600 mL, penicillin 4 million units, 0.4% Phenol red 3.5 mL, adjusted to pH 7.4 ⁇ 7.6 with 10 g/L NaOH.
• lyoprotectant formulated as gelatin 1.5 g, sucrose 12.5 g, double distilled water 100 mL, autoclaved at 115 °C for 15 min, adjusted to pH 7.0 with sterile 1% NaOH. After harvesting the culture, the CCU content was measured, and the lyoprotectant was added in a ratio of 3:2 by volume of the bacteria and the sterilized lyoprotectant. Add the protective agent, mix well, and dispense into an ampoule, 0.5 mL/tube. The ampoules of the prepared strains are placed in a lyophilizer and lyophilized. After lyophilization, the mixture was sealed with nitrogen and stored at -40 °C. One mL of culture was extracted and DNA was extracted for PCR identification.
• the attenuated strain AN306 culture obtained by this method has a titer of up to 10 9 CCU/mL, and the yield is upgraded.
• Example 3 Evaluation of immune protection of intrapulmonary injection of live attenuated vaccine against Mycoplasma hyopneumoniae AN306 strain and comparison with other commercial live vaccines and inactivated vaccines
• Attenuated live vaccine of Mycoplasma hyopneumoniae pneumonia live vaccine AN306 strain dissolve the freeze-dried seedlings with sterile phosphate buffer solution, and prepare a vaccine solution to make the live vaccine titer 2x10 5 CCU/mL. Each animal was intrapulmonarily injected with 0.5 mL, or 10 5 CCU/head.
• the live vaccine of Mycoplasma hyopneumoniae produced by Jilin Zhengye Biological Products Co., Ltd. is a chicken embryo yolk sac tissue containing attenuated rabbit strain of Mycoplasma hyopneumoniae, hereinafter referred to as commercial vaccine 1, according to the vaccine instructions;
• the live vaccine of Mycoplasma hyopneumoniae produced by Jilin Zhengye Biological Products Co., Ltd. is a chicken embryo yolk sac tissue containing attenuated rabbit strain of Mycoplasma hyopneumoniae, hereinafter referred to as commercial vaccine 1, according to the vaccine instructions;
• Commercial vaccine 2 Zhibining, in vitro culture of attenuated strain of Mycoplasma hyopneumoniae 168 strain The following, referred to as commercial vaccine 2, is operated according to the vaccine instructions.
• G1 group was a healthy control group, no immunization was not challenged; G7 group was a challenge control group, and no immunity was only used for challenge.
• the G2-G6 group was the vaccine immunization group.
• the specific grouping and immunization methods are shown in the following table. Animals in the G2-G7 group were intratracheally injected with M. hyopneumoniae for 8 weeks after immunization, and were necropsy after 28 days.
• the Mycoplasma hyopneumoniae pneumonia lesions in the lungs of the animals were scored using the method described in Example 1.
• the results showed that the lung lesions of the live attenuated M. pneumoniae attenuated vaccine AN306 strain were significantly reduced, compared with the control group (statistical method One-way ANOVA), the difference was extremely significant (P ⁇ 0.01), and the mean lesion index was obvious. It is lower than the commercial live vaccine and inactivated vaccine widely used in the market (commodity vaccine 1, P ⁇ 0.01; commercial vaccine 2, P ⁇ 0.05; commercial vaccine 3, P ⁇ 0.01; commercial vaccine 4, P ⁇ 0.01).
• Description Live attenuated vaccine AN306 strain can help immunized animals to effectively resist Mycoplasma hyopneumoniae infection, and the protection is better than the vaccine widely used on the market.
• Table 4 Evaluation of immunoprotection of intrapulmonary injection of live attenuated vaccine against Mycoplasma hyopneumoniae AN306 strain and comparison with other commercial live vaccines and inactivated vaccines
• Attenuated live vaccine of Mycoplasma hyopneumoniae pneumonia attenuated vaccine AN306 strain dissolve the freeze-dried seedlings with vaccine dilution solution, and prepare a vaccine solution to make the live vaccine titer of 10 6 CCU/mL.
• G1 group was healthy control group, no immunization was not challenged
• G2 group was live vaccine aerosol immunization group, aerosol immunization AN306 live vaccine 0.5 mL (5xl0 5 CCU/head), re-immunized 2 weeks after the first immunization, challenged after immunization
• G3 group is a commercial vaccine group, immunizing the commercially available Mycoplasma hyopneumoniae pneumonia inactivated vaccine RespiSure, according to the vaccine instructions Operation, challenge after immunization
• G4 group is the control group of the challenge, not immune only to attack. Animals in G2, G3, and G4 were intratracheally injected with M. hyopneumoniae for 8 weeks after the first immunization, and were examined by necropsy 28 days later. result:
• the Mycoplasma hyopneumoniae pneumonia lesions present in the lungs of the animals were scored using the method described in Example 1, as shown in the table below.
• the results showed that the lung disease of the animal model of attenuated live attenuated Mycoplasma pneumoniae pneumoniae AN306 strain was significantly reduced, which was significantly different from the challenge control group (P ⁇ 0.01), and was significantly lower than the commercial inactivated vaccine (P). ⁇ 0.01). This indicates that the live attenuated vaccine can help immunized animals against M. hyopneumoniae infection by aerosol immunization, and the protection is better than the inactivated vaccine widely used on the market.
• Table 5 Evaluation of immunoprotective capacity of AN306 strain of aerosol-activated live attenuated Mycoplasma pneumoniae pneumonia
• Pulmonary lesion score 1.08 ⁇ 0.27 3.83 ⁇ 2.48 8.25 ⁇ 4.14 20.34 ⁇ 3.45
• Vaccine preparation Attenuated live vaccine of Mycoplasma hyopneumoniae pneumoniae AN306 strain.
• the adjuvant used was a mixture adjuvant containing 5 mg/mL carbomer and 30 mg/mL xanthine polysaccharide formulated in sterile phosphate buffer. Lyophilized vaccine adjuvant solution, the solution is formulated such that the vaccine wherein the live vaccine titer 2.5xl0 5 CCU / mL.
• G1 group was healthy control group, no immunization was not challenged
• G2 group was live vaccine intramuscular injection immunization group, intramuscular injection of AN306 live vaccine 2 mL ( 5xl0 5 CCU/head), re-immunized 2 weeks after the first immunization, challenged after immunization
• G3 group is a commercial vaccine group, immunization is currently used on the market, a wide range of commercially available Mycoplasma hyopneumoniae inactivated vaccine RespiSure, according to the vaccine instructions
• the G4 group was the challenge control group, and the non-immunization only attacked the poison.
• Animals in G2, G3, and G4 were intratracheally injected with M. hyopneumoniae for 8 weeks after the first immunization, and were examined by necropsy 28 days later. result:
• the Mycoplasma hyopneumoniae pneumonia lesions present in the lungs of the animals were scored using the method described in Example 1, as shown in the table below.
• the results showed that the lung lesions of the muscle-injected live attenuated M. pneumoniae attenuated vaccine AN306 strain were significantly reduced, which was significantly different from the challenge control group (P ⁇ 0.01), and significantly lower than the commercial inactivated vaccine (P). ⁇ 0.01).
• P live attenuated vaccine can help immunized animals to resist Mycoplasma hyopneumoniae infection by intramuscular injection, and the protection is better than the inactivated vaccine widely used on the market.
• Table 6 Intramuscular Injection of Liver Attenuated Mycoplasma Pneumoniae Attenuated Live Vaccine Evaluation of Immunoprotection of AN306 Strain Gl G2 G3 G4
• a chitosan-containing vaccine was prepared as follows: chitosan was dissolved in a 1% acetic acid solution to prepare a mother liquor containing 1% chitosan, and the pH was adjusted to 6.5 with a 0.1 M NaOH solution; using 0.1 M NaAc-HAc (pH 6) The solution of .5) was diluted with the above chitosan mother liquor to a final chitosan concentration of 0.2%.
• the fresh culture of Mycoplasma hyopneumoniae AN306 strain was centrifuged at 12,000 rpm for 30 min to collect the cells, and resuspended in 1 mL of 50 mM Na 2 S0 4 to give a titer of 6.66 x 10 6 CCU/mL.
• G1 group was healthy control group, no immunization was not challenged
• G2 group was live vaccine intranasal immunization group, nasal immunization AN306 live vaccine 0.5 mL (5xl0 5 CCU/head), re-immunized 2 weeks after the first immunization, challenged after immunization
• G3 group is a commercial vaccine group, immunization is currently used on the market, a wide range of commercially available Mycoplasma hyopneumoniae inactivated vaccine RespiSure, according to the vaccine instructions Operation, challenge after immunization
• G4 group is the control group of the challenge, not immune only to attack. Animals in G2, G3, and G4 were intratracheally injected with M. hyopneumoniae for 8 weeks after the first immunization, and were examined by necropsy 28 days later. result:
• the Mycoplasma hyopneumoniae pneumonia lesions present in the lungs of the animals were scored using the method described in Example 1, as shown in the table below.
• the results showed that the lung lesions of the live attenuated M. pneumoniae attenuated vaccine AN306 strain were significantly reduced, compared with the control group (P ⁇ 0.01), and significantly lower than the commercial inactivated vaccine (P ⁇ 0.01). ).
• the live attenuated vaccine can help immunized animals against Mycoplasma pneumoniae infection by intranasal immunization, and the protection is better than the inactivated vaccine widely used on the market.
• Example 7 Evaluation of the immunoprotective effect of the live vaccine of Mycoplasma hyopneumoniae AN306 strain and the inactivated vaccine against Haemophilus parasuis
• Serum type 5 H. parasuis XX0306 strain (this strain was isolated by the Institute of Veterinary Medicine of Jiangsu Academy of Agricultural Sciences, and has good immunogenicity. The test proved to be suitable for the preparation of inactivated vaccine against Haemophilus parasuis, and the protective effect is good)
• the adjuvant was formulated with a 5 mg/mL carbomer and immunostimulating complex matrix in a sterile phosphate buffer (component: Quil A 50 ( ⁇ g/mL, cholesterol 10 (Vg/mL, phospholipid 10 (Vg/mL) a mixture of adjuvants.
• the 0.5 mL M. hyopneumoniae fresh culture was mixed with 0.5 mL of serum type 5 H. parasuis inactivated antigen and adjuvant at a volume ratio of 1:1:2 to immunize the animals.
• G1 group was healthy control group, 10 heads, no immunization and no attack, 5 of them were simultaneously necropsy with G2 and G3 animals, and 5 other heads. Simultaneous necropsy with G4 and G5 animals; G2 group was combined with M.
• hyopneumoniae challenge group 5 heads, intramuscular injection of 2 mL of the above-mentioned prepared vaccine (AN306 live vaccine 2.5xl0 5 CCU/mL, serum type 5) Haemophilus parasuis XX0306 strain inactivated antigen 2.5xl0 9 CFU/mL), re-immunized 3 weeks after the first immunization, intratracheal injection of Mycoplasma hyopneumoniae virulent challenge 8 weeks after the first immunization; G3 group was Mycoplasma hyopneumoniae attack Toxicity control group, 5 heads, not immunized, same as G2 group animals; G4 group was combined with M.
• the above-mentioned prepared vaccine AN306 live vaccine 2.5xl0 5 CCU/mL, serum type 5
• Haemophilus parasuis XX0306 strain inactivated antigen 2.5xl0 9 CFU/mL
• parahaemolyticus challenge group 5 heads, intramuscular injection of the above prepared 2 ml vaccine (AN306 live vaccine 2.5 Xl0 5 CCU/mL, serum type 5 Haemophilus parasuis XX0306 strain inactivated antigen 2.5xl0 9 CFU/mL), re-immunized 3 weeks after the first immunization, intraperitoneal injection of Haemophilus parasuis 6 weeks after the first immunization Toxic attack; G5 group was Haemophilus parasuis challenge control group, 5 heads, not immune, with Animals in group G4 also attacked.
• the lung mycoplasma pneumoniae lesions were scored according to the method described in Example 1, and recorded in the table below. The results showed that the combined vaccine could significantly reduce the incidence of lung lesions ( ⁇ 0 ⁇ 01) ).
• the clinical symptoms of Haemophilus parasuis are characterized by decreased appetite, rough hair, redness of the ears and body skin, increased nasal fluid, difficulty in breathing later, swelling of the joints, and lying down.
• the pleural examination showed chest and peritoneal effusion, varying degrees of pleural and peritoneal adhesions, joint swelling, increased mucus in the joint cavity, lung bleeding, and swollen lymph nodes.
• the incidence of Haemophilus parasuis in each group of animals was judged by the above indicators, and the summary is recorded in the following table. The results showed that the immunization of the vaccine could significantly reduce the incidence of Haemophilus parasuis in animals.
• Table 8 Evaluation of immunoprotection of a combination of live vaccine of Mycoplasma hyopneumoniae AN306 and inactivated vaccine of Haemophilus parasuis
• Healthy control group combined vaccine, pig, Mycoplasma pneumoniae, combined vaccine, pig swine, blood stasis, Mycoplasma pneumoniae, challenge control, paraglottic blood test, challenge group, challenge group, bacillus attack group
• Immunization non-immunization, vaccine immunization, non-immunization, immunization with immunization, non-immunization, Mycoplasma hyopneumoniae, no challenge, attack, attack, no attack, no attack, no attack, attack, attack
• Example 8 Evaluation of immunoprotection of live vaccine of Mycoplasma hyopneumoniae AN306 strain and live vaccine of porcine reproductive and respiratory syndrome
• a mixture adjuvant containing 10 mg/mL chitosan and 10 mg/mL levamisole was prepared in sterile phosphate buffer.
• the freeze-dried seedlings of Mycoplasma hyopneumoniae live vaccine AN306 were used to dissolve the freeze-dried M.
• the two vaccine solutions were mixed at a ratio of 3:1 (volume ratio), and the animals were immunized, and each animal was inoculated with 2 mL, that is, one live vaccine of Mycoplasma hyopneumoniae and one live vaccine of Porcine Reproductive and Respiratory Syndrome.
• Animal immunity and attack :
• G1 group was healthy control group, 10 heads, no immunization and no attack, 5 of them were simultaneously necropsy with G2 and G3 animals, and 5 other heads. Simultaneous necropsy with G4 and G5 animals; G2 group was combined with M.
• hyopneumoniae challenge group 5 heads, intramuscularly immunized 2 mL of the above-mentioned prepared vaccine, re-immunized 2 weeks after the first immunization, 8 weeks after the first immunization
• G3 group was a Mycoplasma hyopneumoniae challenge control group, 5 heads, not immunized, same as G2 group of animals
• G4 group was combined with vaccine against pig reproductive and respiratory syndrome virus attack Toxic group, 5 heads, intramuscularly immunized 2mL of the above-mentioned prepared vaccine, re-immunized 2 weeks after the first immunization, 6 months after the first immunization, intramuscular injection of porcine reproductive and respiratory syndrome virus virulent challenge
• G5 group Pig Breeding and Respiratory Syndrome virus challenge control group, 5, not immune, and the same as G4 animals.
• the lung mycoplasma pneumoniae lesions were scored according to the method described in Example 1, and recorded in the table below. The results showed that the combined vaccine could significantly reduce the incidence of lung lesions ( ⁇ 0 ⁇ 01) ).
• the criteria for the diseased pigs were: high fever above 41 °C for more than 3 days, decreased mental and appetite, respiratory conjunctivitis and respiratory symptoms such as cough and gasp, and death.
• the necropsy showed a flaky consolidation.
• the results showed that all the animals in the control group of the porcine reproductive and respiratory syndrome virus challenged, 2 of which died, and the animals in the combined vaccine group had no obvious symptoms and all survived.
• Table 9 Evaluation of the immunoprotective effect of the live vaccine of Mycoplasma hyopneumoniae AN306 strain and live vaccine of porcine reproductive and respiratory syndrome
• Healthy control group combined vaccine, pig, Mycoplasma pneumoniae, vaccine vaccine, pig breeding and respiratory, Mycoplasma pneumoniae, challenge control, pig breeding and sucking syndrome, attack group, respiratory syndrome, poison challenge, virus attack, virus group
• Immunization, non-immunization, immunization, immunization, immunization, immunization, immunization, immunization Mycoplasma pneumoniae does not attack and attack, does not attack, does not attack, breeds pigs, breeds and breaths, does not attack, does not attack, does not attack, does not attack

Priority Applications (3)

Applications Claiming Priority (1)

Publications (1)

Family

ID=53680680

Family Applications (1)

Country Status (3)

Cited By (3)

Families Citing this family (7)

Citations (4)

• 2014
  • 2014-01-26 WO PCT/CN2014/071506 patent/WO2015109578A1/zh active Application Filing
  • 2014-01-26 EP EP14879895.2A patent/EP3098301B1/en active Active
  • 2014-01-26 US US15/113,909 patent/US9849168B2/en active Active

Patent Citations (4)

Also Published As

Similar Documents

Legal Events